Objective To investigate the underlying mechanisms of endothelial cell spreading. Methods Human umbilical vein endothelial cells （HUVECs） were plated onto fibronectin （FN）-coated plates and in some experiments the cells were pre-treated with phosphatidyl inositol 3-kinase （PI3K）-Akt signaling pathway inhibitors and integrin inhibitor. Then the cell areas and the activation of Akt were determined. Results When HUVECs were grown on FN，the cell areas were gradually increased in a time-dependent manner and reached a plateau in 120 min. The phosphorylation of Akt significantly increased with FN stimulation for 15 min and reached the peak in 60 min. However，the PI3K-Akt signaling pathway inhibitors，LY294002 and wortmannin significantly prevented FN-induced endothelial cell spreading. Moreover，the blockade of integrin with RDGS peptide significantly inhibited Akt activation and cell spreading. Conclusion Integrin-dependent Akt activation might be crucial for FN-induced endothelial cell spreading.